Glioma cells deficient in urokinase plaminogen activator receptor expression are susceptible to tumor necrosis factor-alpha-related apoptosis-inducing ligand-induced apoptosis.

PURPOSE: The urokinase plasminogen activation system comprises the ligand urokinase plasminogen activator and the receptor urokinase plasminogen activator receptor (uPAR), which play an important role in the activation of matrix-degrading enzymes that enhance the invasion of cancer cells. Earlier studies have indicated that SNB19 glioblastoma cells expressing antisense uPAR constructs lose their invasive properties when injected in vivo. Additional observations indicated that injected antisense uPAR:SNB19 cells were being lost through apoptotic elimination. EXPERIMENTAL DESIGN: SNB19, Vector, and SNB19:asuPAR were analyzed to determine cytotoxicity of tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL), receptor expression, and underlying signaling pathways using flow cytometry, immunohistochemistry, RNase protection assay, and c-Jun-NH(2)-terminal kinase activity. RESULTS: This study elucidated the susceptibility of antisense uPAR:SNB19 cells to TRAIL under certain experimental conditions in vitro. These uPAR-deficient transfected cells had higher levels of the TRAIL receptors DR4 and DR5 than did the control and vector population as detected by flow cytometry. An RNase protection assay confirmed the elevation of DR4 and DR5 mRNA in the antisense uPAR cells. CONCLUSIONS: These findings provide preliminary evidence of a link between TRAIL-induced apoptosis and cell cycle progression in antisense uPAR:SNB 19 cells.

A novel function of tissue factor pathway inhibitor-2 (TFPI-2) in human glioma invasion.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase-type plasminogen activator, tissue plasminogen activator, or thrombin. Preliminary findings in our laboratory suggested that the expression of TFPI-2 is downregulated or lost during tumor progression in human gliomas. To investigate the role of TFPI-2 in the invasiveness of brain tumors, we stably transfected the human high-grade glioma cell line SNB19 and the human low-grade glioma cell line Hs683 with a vector capable of expressing a transcript complementary to the full-length TFPI-2 mRNA in either sense (0.7 kb) or antisense (1 kb) orientations. Parental cells and stably transfected cell lines were analysed for TFPI-2 protein by Western blotting and for TFPI-2 mRNA by Northern blotting. The levels of TFPI-2 protein and mRNA were higher in the sense clones (SNB19) and decreased in the antisense (Hs683) clones than in the corresponding parental and vector controls. In spheroid and matrigel invasion assays, the SNB19 parental cells were highly invasive, but the sense-transfected SNB-19 clones were much less invasive; the antisense-transfected Hs683 clones were more invasive than their parental and vector controls. After intracerebral injection in mice, the sense-transfected SNB19 clones were less able to form tumors than were their parental and vector controls, and the antisense-Hs683 clones but not the parental or vector controls formed small tumors. This is the first study to demonstrate that down- or upregulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas.

Down-regulation of integrin alpha(v)beta(3) expression and integrin-mediated signaling in glioma cells by adenovirus-mediated transfer of antisense urokinase-type plasminogen activator receptor (uPAR) and sense p16 genes.

Interaction between the extracellular matrix and integrin receptors on cell surfaces leads not only to cell adhesion but also to intracellular signaling events that affect cell migration, proliferation, and survival. The vitronectin receptor alpha(v)beta(3) integrin is of key importance in glioma cell biology. The expression of urokinase-type plasminogen activator receptor (uPAR) was recently shown to co-regulate with the expression of alpha(v)beta(3) integrin. Moreover, restoration of the p16 protein in glioma cells inhibits the alpha(v)beta(3) integrin-mediated spreading of those cells on vitronectin. Thus we hypothesized that adenovirus-mediated down-regulation of uPAR and overexpression of p16 might down-regulate the expression of alpha(v)beta(3) integrin and the integrin-mediated signaling in glioma cells, thereby defeating the malignant phenotype. In this study, we used replication-deficient adenovirus vectors that contain either a uPAR antisense expression cassette (Ad-uPAR) or wild-type p16 cDNA (Ad-p16) and a bicistronic adenovirus construct in which both the uPAR antisense and p16 sense expression cassettes (Ad-uPAR/p16) are inserted in the E1-deleted region of the vector. Infecting the malignant glioma cell line SNB19 with Ad-uPAR, Ad-p16, or Ad-uPAR/p16 in the presence of vitronectin resulted in decreased alpha(v)beta(3) integrin expression and integrin-mediated biological effects, including adhesion, migration, proliferation, and survival Our results support the therapeutic potential of simultaneously targeting uPAR and p16 in the treatment of gliomas.

Stable transfection of urokinase-type plasminogen activator antisense construct modulates invasion of human glioblastoma cells.

The diffuse and extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modifications of the proteolysis of extracellular matrix components. A key molecule in regulating plasminogen-mediated extracellular proteolysis is the urokinase-type plasminogen activator (uPA). To investigate the role of uPA in the invasive process of brain tumors, we stably transfected a human glioblastoma cell line SNB19 with a vector capable of expressing an antisense transcript complementary to the 1020 bases at the 3' end of the uPA cDNA. Parental, vector-, and antisense construct-stably transfected cell lines were analyzed for uPA mRNA transcript by Northern blot analysis, for uPA enzyme activity by zymography, and for uPA protein levels by Western blotting. The levels of uPA mRNA, protein, and enzyme activities were significantly lower in antisense clones than in parental and vector controls. Radioreceptor binding studies demonstrated that uPA receptor levels remained the same in parental, vector-, and antisense-transfected cells. The antisense-transfected cells showed a markedly lower level of invasion in the Matrigel invasion assays, and their spheroids failed to invade the fetal rat brain aggregates in the coculture system. Green fluorescent protein (GFP) expressing parental and antisense transfectants was generated for detection in mouse brain tissue without any posttreatment. Intracerebral injection of antisense stable transfectants significantly reduced tumor formation compared with that in controls. Our results suggested that down-regulation of uPA expression may be a feasible approach to reducing the malignancy and invasiveness of glial tumors.

Elevated levels of cathepsin B in human glioblastoma cell lines.

Degradation of the extracellular matrix is a prerequisite for the invasive phenotype in glioma cells. Several proteases released by invading tumor cells seem to participate in the focal degradation of extracellular matrix proteins. Using enzymatic assays, Western blotting, and Northern blotting techniques, we investigated whether cathepsin B level was associated with malignant grade in seven human glioma cell lines. Cathepsin B activity and protein content levels were higher in glioblastoma cell lines than in anaplastic astrocytoma or low-grade glioma cell lines. Cathepsin B transcripts were overexpressed in glioblastoma cell lines relative to their expression in anaplastic astrocytoma and low-grade glioma cell lines. Cathepsin B promoter activity and amount of SP-1 complexes were much higher in glioblastoma cell lines than in anaplastic astrocytoma or low-grade glioma cell lines. Finally, E-64, an inhibitor of cathepsin B, inhibited both cathepsin B enzymatic activity and the invasiveness of glioblastoma cell lines. These results strongly support a role for cathepsin B in glioblastoma cell lines and suggest that inhibition of cathepsin B activity may be proven useful in cancer therapy.

A novel role of tissue factor pathway inhibitor-2 in apoptosis of malignant human gliomas.

Tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa serine protease inhibitor found at high levels in extracellular matrix. Recombinant human TFPI-2 has recently been shown to be a strong inhibitor of trypsin, plasmin, plasma kallikrein, and factor XIa amidolytic activity. Earlier studies in our laboratory showed that the expression of TFPI-2 is lost during tumor progression in human gliomas. We stably transfected this protease inhibitor in multiform glioblastoma cell line (SNB-19) and in low-grade glioma cell line (Hs683) in sense and antisense orientation respectively. This confirmed that the upregulation/down-regulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas both in vitro and in vivo models. Collectively, these results suggested an idea to determine whether TFPI-2 is necessary for cell survival and inhibition of tumor formation in nude mice, due to apoptosis of intracerebrally injected SNB-19 cells. In the present study we determined p-ERK levels and found that they are decreased in TFPI-2 over-expressed clones (SNB-19) and increased in TFPI-2 down-regulated clones (Hs683). We also checked the levels of BAX/BCl-2, caspases (for e.g., 9, 7, 3, 8), PARP, cytochrome-c and Apaf-1. Moreover, the increase of apoptosis in vitro is associated with increased and decreased expression of apoptotic protein BAX in sense clones (SNB-19) and antisense clones (Hs683) respectively, when compared to controls and vice versa with Bcl-2 the anti-apoptotic protein. Caspases (9, 7 and 3), cytochrome-c, Apaf-1 and PARP levels are increased in SNB-19 and decreased in Hs683. Caspase 8 was not expressed in either cell line. Caspases 9 and 3 activity assay revealed higher activity in sense clones (SNB-19) but lesser in antisense clones (Hs683) compared to controls. This is the first report of TFPI-2 playing a novel role in cell survival in human gliomas.

Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells.

Increases in abundance of cathepsin B transcript and protein correlate with increases in tumor grade and alterations in subcellular localization and activity of cathepsin B. The enzyme is able to degrade the components of the extracellular matrix (ECM) and activate other proteases capable of degrading ECM. To investigate the role played by this protease in the invasion of brain tumor cells, we transfected SNB19 human glioblastoma cells with a plasmid containing cathepsin B cDNA in antisense orientation. Control cells were transfected with vector alone. Clones expressing antisense cathepsin B cDNA exhibited significant reductions in cathepsin B mRNA, enzyme activity and protein compared to controls. Matrigel Invasion assay showed that the antisense-transfected cells had a markedly diminished invasiveness compared with controls. When tumor spheroids containing antisense transfected SNB19 cells expressing reduced cathepsin B were co-cultured with fetal rat brain aggregates, invasion of fetal rat brain aggregates was significantly reduced. Green Fluorescent Protein (GFP) expressing parental cells and antisense transfectants were generated for detection in mouse brain tissue without any post-chemical treatment. Intracerebral injection of SNB19 stable antisense transfectants resulted in reduced tumor formation in nude mice. These results strongly support a role for cathepsin B in the invasiveness of human glioblastoma cells and suggest cathepsin B antisense may prove useful in cancer therapy.

Adenovirus-mediated antisense urokinase-type plasminogen activator receptor gene transfer reduces tumor cell invasion and metastasis in non-small cell lung cancer cell lines.

The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the proteolytic cascade involved in the metastasis of lung and other cancers. We report that the reduction in uPAR levels produced by an antisense strategy using an adenovirus construct (Ad-uPAR) in H1299 cells, an invasive human lung cancer cell line that produces high levels of uPAR, resulted in a decrease of uPAR levels to 80-90% of those seen in cells infected with mock or adenovirus (Ad)-cytomegalovirus vector controls. In addition, increasing the multiplicity of infection from 25 to 200 caused a corresponding decrease in the level of uPAR protein within 5 days of treatment, as shown by Western blot analysis. Furthermore, the in vitro translation of total RNA levels of Ad-uPAR-infected H1299 cells in a rabbit reticulocyte lysate system caused a 50-70% decrease in uPAR immunoprecipitate in Ad-uPAR-infected cells relative to the levels in cells of mock and vector controls. The Matrigel invasion assay showed the invasion of H1299 cells and A549 cells infected with Ad-uPAR to be decreased by 70% relative to mock- and vector-infected controls. Infection of tumor cells with Ad-uPAR before implantation significantly reduced the incidence of lung metastasis by 85% as compared with the control virus-infected cells injected into nude mice through the tail vein. Our collective results show that the uPAR system is a potential target of treatment for lung cancers.

The prognosis of children with hydrocephalus and congenital heart disease.

The association of congenital hydrocephalus and heart disease in children is infrequent, but may present considerable dilemmas in management. This report describes the treatment and prognosis of 11 children with both clinical problems. There were 5 males and 6 females. Hydrocephalus occurred following aqueductal stenosis in 5 children and the Dandy-Walker malformation in 3. Three children were diagnosed with idiopathic hydrocephalus. Ten children underwent cerebrospinal fluid diversion procedures for control of hydrocephalus. Five children received pharmacological therapy for cardiac disease; 4 children required surgical correction. Two children died from medical conditions; 2 families declined treatment. Follow-up from 2 to 7 years in the remaining 7 children demonstrated moderate or severe neurodevelopmental disability in 5. One child at 2 years of age showed borderline developmental disability while 1 child is developing normally at 10 years of age. Overall the occurrence of symptomatic hydrocephalus and heart disease in the perinatal period resulted in mortality or neurodevelopmental disability in 9/11 children.

Radiation-induced cavernous angioma mimicking metastatic disease.

Patients with carcinoma of the lung typically have a limited life expectancy especially after developing metastatic disease in the brain. New enhancing lesions in the brain are usually felt to represent new areas of metastasis. Recently, there have been several case reports of cavernous angiomas appearing years after radiation to the brain, typically in children. We present a case of a 41-year-old gentleman with carcinoma of the lung with metastasis to the brain who received postoperative radiation. Five-and-a-half years later he presented with a new enhancing lesion of the brain with surrounding vasogenic oedema, thought to represent a metastatic tumour. It proved is the a radiation-induced cavernous angioma.

Downregulation of urokinase-type plasminogen activator receptor (uPAR) induces caspase-mediated cell death in human glioblastoma cells.

Urokinase-type plasminogen activator receptors (uPARs) play an important role in tumor invasion by localizing degradative enzymes at the invasive zone. Our previous studies with human glioblastoma cell line SNB19 expressing AS-uPAR stable tranfectant lose their invasive properties when injected in vivo. The aim of the present study is to investigate whether the inhibition of tumor formation is due to apoptosis. Apoptosis is a highly conserved cell suicide program essential for development and tissue homeostasis of all metazoan organisms. Key to the apoptotic program is a family of cystein proteases termed caspases, which are important for execution of apoptosis by cleavage of essential cellular proteins. We found loss of mitochondrial transmembrane potential, release of cytochrome C from mitochondria and subsequent activation of Caspase-9 in SNB 19 AS-uPAR cells compared to parental and vector controls. Our results indicate that suppression of uPAR results in apoptosis and suggest that Caspase-9 dependent apoptosis plays an important role in SNB19 AS-uPAR-induced apoptosis.

Modulation of endothelial cell morphogenesis in vitro by MMP-9 during glial-endothelial cell interactions.

The purpose of this study was to investigate the roles of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the formation of capillary structures by human brain microvascular endothelial cells cocultured with SNB19 glioblastoma cells. Unstimulated cocultures did not form capillaries and produce MMP-9 but stimulation with the protein kinase C (PKC) activator 4-phorbol-12-myristate 13-acetate (PMA) produced MMP-9 and capillary networks. Addition of recombinant MMP-9 increased capillary formation. Anti-MMP-9 antibodies, TIMP-1, the synthetic MMPs inhibitor Batimastat (BB-94), and the PKC inhibitor calphostin-C all reduced MMP-9 activity and capillary network formation in these cocultures. Cytochalasin-D in the presence of PMA suppressed MMP-9 expression and capillary formation, but colchicine-B had no such effect. Finally, PMA-induced MMP-9 expression and capillary formation were inhibited by the MEKK-specific inhibitor PD98059. These results suggest that MMP-9 is important in endothelial cell morphogenesis and the formation of capillaries in glial/endothelial cocultures in vitro.

Far-lateral disc herniations: the results of conservative management.

The far-lateral herniated lumbar disc has become increasingly recognized as a cause for low back pain and lumbar radiculopathy as well as for "failed back syndrome" in certain improperly diagnosed cases. Several authors have reported that the majority of patients show poor response to conservative measures. To better understand the natural history, we performed a retrospective review of all lumbar herniated discs during a 3-year period, collecting 16 patients with 17 far-lateral disc herniations. All displayed radicular pain in the distribution of the root exiting at the same level as the herniated disc, with or without associated back pain. Twelve of the 17 disc herniations responded to conservative measures and had complete resolution of their radicular pain at follow-up. Also, at long-term follow-up, essentially all patients had experienced satisfactory subjective resolution of their weakness or sensory complaints. Five patients required surgery because of intractable pain despite conservative measures. Although our series for far-lateral disc herniations is small, we found that conservative measures do afford a relatively high nonoperative success rate of approximately 71%. This is in contrast to earlier implied or stated opinions indicating a low rate of successful nonoperative management as low as 10% in one series.

Bilateral enhancing thalamic lesions in a 10 year old boy: case report.

A young boy presented with monoparesis of the left arm. MRI disclosed bilateral enhancing thalamic lesions. Biopsy results and subsequent clinical history were most compatible with postinfectious or acute disseminated encephalomyelitis. This represents one of the first cases of acute disseminated encephalomyelitis affecting the thalami, established by biopsy. This uncommon disease entity is reviewed and how it may affect the deep grey matter is described.

The natural history and growth rate of asymptomatic meningiomas: a review of 60 patients.

Little is known about the natural history and growth rate of asymptomatic meningiomas. To better delineate this problem, the authors reviewed the clinical records and imaging studies of the last 60 patients diagnosed with asymptomatic meningiomas at their institution. There were 45 women and 15 men, whose ages ranged from 38 to 84 years, with a mean age of 66 years. The most common tumor location was convexity (25 patients), but virtually all locations were represented. Three patients were lost to follow up. The average clinical follow-up review of the remaining 57 patients was 32 months (range 6 months to 15 years). None of the patients became symptomatic from an enlarging tumor during their follow-up period. Typically, once a meningioma was diagnosed, follow-up scans were obtained at 3 months, 9 months, and then yearly or every other year thereafter. Forty-five patients underwent follow-up scans, with comparison of tumor size to that found on the initial scan, over a period ranging from 3 months to 15 years. Thirty-five patients have shown no growth in their tumor size, with an average imaging follow up of 29 months (range 3-72 months). Ten patients have shown tumor growth calculated as an increase in the maximum diameter of the tumor. This growth ranged from 0.2 cm over 180 months to 1 cm over 12 months, with an average of 0.24 cm per year. Average imaging follow up for these patients was 47 months (range 6 months to 15 years). The authors conclude that patients with asymptomatic meningiomas need close clinical and radiological follow up to rule out other disease processes and to rule out rapidly enlarging tumors. Although the average follow-up time was short, the vast majority of these tumors appeared to show minimal or no growth over periods of time measured in years. With modern noninvasive imaging techniques, these tumors can be safely observed until they enlarge significantly or become symptomatic.

The use of PET in evaluating patients with primary brain tumours: is it useful?

During an 18 month period 39 patients were evaluated with [18F] fluorodeoxyglucose-PET (FDG-PET) for primary brain tumours. These included patients with suspected newly diagnosed tumours and patients with known tumours who were being evaluated for possible recurrence or increasing tumour grade. Scans were performed on a 951-31 Siemen's PET scanner with 4 mm resolution. Scanning time was about 20 minutes per patient. All patients had undergone recent cerebral MRI. These patients were divided into two groups. In the first group (30) MRI and PET concurred on the diagnosis. The second group (nine) comprised those where the interpretation of MRI and PET was different or there was a question of the diagnosis on MRI. This group comprised three patients in whom MRI suggested recurrent tumour and PET inaccurately suggested radiation necrosis; two patients with newly diagnosed enhancing lesions on MRI in whom PET was useful in distinguishing strokes from tumour; two patients with prior gliomas with new enhancing isolated lesions on MRI in whom PET scan accurately depicted radiation necrosis; and two patients with newly diagnosed enhancing lesions on MRI in whom PET scan was helpful in distinguishing multiple sclerosis from tumour in one but not in the other. Therefore, of the 39 patients, PET was helpful in five in distinguishing tumour from other disease processes; but, in so far as influencing treatment, it seemed helpful in only two. Thus PET seems to be of limited value as an aid to evaluating and treating patients with suspected or known primary brain tumours.

A risk/benefit analysis of spinal manipulation therapy for relief of lumbar or cervical pain.

Approximately 12 million Americans undergo spinal manipulation therapy (SMT) every year. Renewed interest in this method requires an analysis of its reported risks and possible benefits. This review describes two patients with spinal cord injuries associated with SMT and establishes the risk/benefit ratios for patients with lumbar or cervical pain. The first case is a man who underwent SMT for recurrent sciatica 4 years after chemonucleolysis. During therapy, he developed bilateral sciatica with urinary hesitancy. After self-referral, myelography demonstrated a total block; he underwent urgent discectomy with an excellent result 3 months after surgery. The second patient with an indwelling Broviac catheter and a history of lumbar osteomyelitis underwent SMT for neck pain. Therapy continued for 3 weeks despite the development of severe quadriparesis. After self-referral, he underwent an urgent anterior cervical decompression and removal of necrotic bone and an epidural abscess with partial neurological recovery. An analysis of these cases and 138 cases reported in the literature demonstrates six risk factors associated with complications of SMT. These include misdiagnosis, failure to recognize the onset or progression of neurological signs or symptoms, improper technique, SMT performed in the presence of a coagulation disorder or herniated nucleus pulposus, and manipulation of the cervical spine. Clinical trials of SMT have been summarized in several recent articles.(ABSTRACT TRUNCATED AT 250 WORDS)